The brush border (BB) Na/H exchanger NHE3 is rapidly up and down regulated as part of digestion, with prolonged inhibition contributing to the pathophysiology of diarrhea. NHE3 exists in the BB in large, multiprotein complexes of varying size. In these complexes, NHE3 associates with 4 BB PDZ domain containing proteins, NHERF1, NHERF2, PDZK1 and IKEPP. The BB PDZ proteins are in somewhat different locations in Na absorptive cells, although each has a BB component. This project will test the hypotheses that acute NHE3 regulation in intestinal BB requires its presence in large multiprotein complexes scaffolded by these BB PDZ proteins; and that these complexes are dynamic and change in their location and makeup as part of NHE3 regulation. Proposed studies will knock down (shRNAi in Caco-2 cells) and out (gene targeting in mice) each of these BB PDZ proteins alone and in combinations. Physiologic and pathophysiologic regulation of NHE3 in mouse ileum and Caco-2 cells will be evaluated using the Ussing chamber/voltage clamp approach and two-photon microscopy with SNARF-4F to measure intracellular pH. Basal and acutely stimulated and inhibited NHE3 activity will be examined, with determination whether trafficking of NHE3 is affected; what size complexes in the BB NHE3 and the BB PDZ proteins are in, using sucrose density gradient centrifugation, under these conditions; and whether NHE3/NHERF proteins change their direct association as part of NHE3 regulation as assessed by FRET. Knockout/down models of the 4 BB PDZ proteins (NHERF1, NHERF2 already on hand and PDZK1 is available) will be evaluated to understand their contribution to NHE3 regulation. NHE3 associates with the cytoskeleton directly by ezrin binding and indirectly by binding to ezrin via binding NHERF1, NHERF2 and PDZK1. Point mutations of NHE3 have been identified which separately eliminate this direct and indirect ezrin binding. These mutants will be expressed in Caco-2 cells and effects determined on NHE3 BB localization and on NHE3 basal and regulated activity, NHE3 complex formation and associating proteins, as well as on NHE3 mobility in the BB as assessed by FRAP. These studies will provide insight into how salt is absorbed normally in the intestine and becomes abnormal in diarrheal diseases.